Nrf2 mediates hypoxia-inducible HIF1α activation in kidney tubular epithelial cells

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2^-/-) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2^-/- RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2^-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2^-/- RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI.

Keywords: acute kidney injury; hypoxia-inducible factor-1α; ischemia-reperfusion; mitochondria; nuclear factor erythroid 2-related factor 2.

Graphical abstract

Figure 1.

HIF1α regulates Nrf2 target HMOX1 induction by hypoxia. A: HMOX1 levels in HK2 cells treated with HIF1 pathway inhibitor digoxin (10 µM) or DMSO and exposed to RA or 12-h hypoxia (0hR) and reoxygenated for 6 h (6hR). Cellular extracts were prepared and immunoblotted with HIF1α and HMOX1 antibodies using β-actin as reference. B: primary RTECs were isolated from WT and Nrf2^–/– mice and cultured for 6–7 days and then treated with 200 µM cobalt (II) chloride (CoCl₂) (60818, Sigma) for 12 h. Hmox1 expression was analyzed by qRT-PCR. * Versus vehicle; § versus WT. Two-way ANOVA was used to calculate the effects of CoCl₂ on Hmox1 expression (n = 3 for vehicle; and n = 4 for CoCl₂-treated groups). HIF1α, hypoxia-inducible factor 1α; HMOX1, heme oxygenase 1, HK2, human immortalized renal tubular epithelial cells; Nrf2, Nuclear factor erythroid 2-related factor 2; RA, room air; RTECs, primary murine kidney tubular epithelial cells; WT, wild type.

Figure 2.

HIF1α target gene expression induced by hypoxia is impaired in Nrf2^–/– RTECs. WT and Nrf2^–/– RTECs exposed to 2 h (A) or 12 h (B) hypoxia and then subjected to reoxygenation for 6 h. HIF1α putative targets Vegf1, Epo, Glut1, Pdk1, and Pgk1 mRNA expression were analyzed by qRT-PCR analysis. Two-way ANOVA with Tukey’s multiple-comparisons test was used to calculate the effects of hypoxia or reoxygenation on target gene expression (n = 6–8). *Versus room air; §versus WT counterparts. Epo, erythropoietin; Glut1, glucose transporter 1; HIF1α, hypoxia-inducible factor 1α; Nrf2^–/–, Nrf2-deficient; Pdk1, pyruvate dehydrogenase kinase 1; Pgk1, phosphoglycerate kinase 1; RTECs, primary murine kidney tubular epithelial cells; WT, wild type.

Figure 3.

Nrf2 deficiency impairs hypoxia-stimulated HIF1α activation. A: primary cultured RTECs isolated from WT and Nrf2^–/– mice were exposed to hypoxia for 0 h, 0.5 h, 1 h, and 2 h. B: WT and Nrf2^–/– RTECs were exposed to RA or acute hypoxia for 2 h (0hR) and subsequently reoxygenated for 6 h (6hR). C: WT and Nrf2^–/– RTECs exposed to chronic (12 h) hypoxia and reoxygenated for 6 h (6hR). Cells were lysed and immunoblotted with HIF1α antibody β-actin antibodies. A representative blot of two independent experiments is shown. HIF1α, hypoxia-inducible factor 1α; Nrf2, nuclear factor erythroid 2-related factor 2; Nrf2^–/–, Nrf2-deficient; RTECs, primary murine kidney tubular epithelial cells; WT, wild type.

Figure 4.

Hypoxia-stimulated PHD expression is regulated by Nrf2. WT and Nrf2^–/– RTECs were exposed to 2-h or 12-h hypoxia and reoxygenated for 6 h. Phd1/2/3 mRNA expression was analyzed by qRT-PCR. A: mRNA expression induced by 2-h hypoxia and followed by reoxygenation. B: mRNA levels induced by 12-h hypoxia and followed by reoxygenation. Two-way ANOVA with Tukey’s multiple-comparisons test was used to calculate the effects of hypoxia or reoxygenation on target gene expression (n = 6–8). *Versus room air; §versus WT counterparts. Nrf2, nuclear factor erythroid 2-related factor 2; Nrf2^–/–, Nrf2-deficient; PHD, prolyl 4-hydroxylase; RTECs, primary murine kidney tubular epithelial cells; WT, wild type.

Figure 5.

Nrf2 positively regulates HIF1α mRNA expression. WT and Nrf2^–/– RTECs exposed to 2-h or 12-h hypoxia and/or subjected for reoxygenation. HIF1α mRNA expression was analyzed by qRT-PCR. A: expression levels in 2-h hypoxia and reoxygenation for 6 h. B: expression levels in 12-h hypoxia and reoxygenation for 6 h. Two-way ANOVA with Tukey’s multiple-comparisons test was used to calculate the effects of hypoxia or reoxygenation on target gene expression (n = 6–8). *Versus room air; §versus WT counterparts. HIF1α, hypoxia-inducible factor 1α; Nrf2, nuclear factor erythroid 2-related factor 2; Nrf2^–/–, Nrf2-deficient; RTECs, primary murine kidney tubular epithelial cells; WT, wild type.

Figure 6.

Nrf2 binds to the endogenous HIF1α promoter in vivo. HK2 cells exposed to room air (RA), 2-h/12-h hypoxia (0hR), and hypoxia-reoxygenation for 1 h (1hR), 3 h (3hR) or 6 h (6hR). ChIP assays were performed using anti-Nrf2 antibodies. IgG antibodies were used as negative controls. A: schema of human HIF1α promoter with a transcriptional start site and Sp1 sites (11, 17). Position of putative Nrf2-binding sites, AREs, and primer 1 and primer 2 used for ChIP assay are indicated. Bottom represents murine HIF1α promoter. B: Nrf2 binding to the human HIF1α promoter in HK2 cells exposed to 2-h hypoxia and hypoxia-reoxygenation. C: Nrf2 binding to the human HIF1α promoter in HK2 cells exposed to 12-h hypoxia and hypoxia-reoxygenation. ChIP assays performed with IgG antibodies showed undetectable levels of promoter amplification (not shown). Student’s t test was used to calculate the effects of hypoxia or reoxygenation on Nrf2 binding compared with RA control (n = 8–9) or hypoxia (0hR), respectively. *Versus RA; §versus 0hR. AREs, antioxidant response elements; ChIP, chromatin immunoprecipitation; HIF1α, hypoxia-inducible factor 1α; HK2, human immortalized tubular epithelial cells; Nrf2, Nuclear factor erythroid 2-related factor 2; RA, room air.

Figure 7.

Hypoxia downregulates Nrf2 levels. A: HK2 cells were exposed to hypoxia for 30, 60, 90, or 120 mins (0hR) followed by reoxygenation for 3 h (3hR). B: HK2 cells were exposed to room air or hypoxia for 15, 30, or 60 mins. Kinetics of Nrf2 restoration during reoxygenation following acute hypoxia (C) and chronic hypoxia (D). Cells were lysed and immunoblotted with Nrf2 and β-actin antibody. A representative blot of two independent experiments is shown. HK2, human immortalized tubular epithelial cells; Nrf2, Nuclear factor erythroid 2-related factor 2.

Figure 8.

The effects of proteasome and protein kinase inhibition on Nrf2 levels in hypoxia. HK2 cells were exposed to room air or hypoxia for 60 min in the presence of vehicle (DMSO) (D2650, Sigma), 10 µM MG132 (M7449, Sigma), 10 µM cycloheximide (C4859, Sigma) (A), or 10 µM bisindolylmaleimide I (BIM I) (203290, Calbiochem) (B). Cell extracts were prepared and probed with Nrf2 and β-actin antibodies. A representative blot of two independent experiments is shown. HK2, human immortalized renal tubular epithelial cells; Nrf2, Nuclear factor erythroid 2-related factor 2.

Figure 9.

Mitochondrial complex I mediates hypoxia-induced Nrf2 downregulation and HIF1α target gene expression. A: HK2 cells treated with 10 µM rotenone (557368, Calbiochem) or 10 µM antimycin A (A8674, Sigma) or DMSO for 1 h were exposed to room air or hypoxia for 1 h, cell lysates prepared, and immunoblotted with β-actin and Nrf2 antibodies. A representative blot of at least two independent experiments is shown. B: HK2 cells treated with DMSO or 10 µM rotenone for 6 h, RNA isolated, and gene expression was analyzed by qRT-PCR. Student’s t test was used to calculate the effect of rotenone on gene expression (n = 6). * Versus DMSO. C: the effects of rotenone on HIF1α levels. Cellular extracts treated with rotenone were probed with HIF1α and β-actin antibodies. D: schema of mitochondria-mediated Nrf2 destabilization and HIF1α stabilization in hypoxia and vice versa in reoxygenation. Basal level Nrf2 binds to the promoter and positively regulates constitutive HIF1α mRNA expression (left). Optimal level HIF1α stabilization and its target metabolic gene induction in hypoxia is Nrf2 dependent. Concurrent with HIF1α activation, Nrf2 levels are downregulated in hypoxia via mitochondrial complex I (middle). Reoxygenation destabilizes HIF1α and increases Nrf2 levels leading to antioxidant enzyme expression and mitigation of excessive ROS occurs. In addition, Nrf2 maintains basal-level HIF1α gene expression (right). HIF1α, hypoxia-inducible factor 1α; HK2, human immortalized renal tubular epithelial cells; Nrf2, Nuclear factor erythroid 2-related factor 2; RNA, ribonucleic acid.